ABSTRACT
BACKGROUND: Homologous recombination repair (HRR) pathway deficiencies have significant implications for cancer predisposition and treatment strategies. Improved quantitative methods for functionally characterizing these deficiencies are required to accurately identify patients at risk of developing cancer and to identify mechanisms of drug resistance or sensitivity. METHODS: Flow cytometry-based single cell network profiling (SCNP) was used to measure drug-induced activation of DNA damage response (DDR) proteins in cell lines with defined HRR pathway mutations (including ATM-/-, ATM+/-, BRCA1+/-, BRCA2-/-) and in primary acute myeloid leukemia (AML) samples. Both non-homologous end joining (NHEJ) and HRR pathways were examined by measuring changes in intracellular readouts (including p-H2AX, p-ATM, p-DNA-PKcs, p-53BP1, p-RPA2/32, p-BRCA1, p-p53, and p21) in response to exposure to mechanistically distinct genotoxins. The cell cycle S/G2/M phase CyclinA2 marker was used to normalize for proliferation rates. RESULTS: Etoposide induced proliferation-independent DNA damage and activation of multiple DDR proteins in primary AML cells and ATM +/+but not ATM -/- cell lines. Treatment with the PARPi AZD2281 +/- temozolomide induced DNA damage in CyclinA2+ cells in both primary AML cells and cell lines and distngiushed cell lines deficient (BRCA2-/-) or impaired (BRCA1+/-) in HRR activity from BRCA1+/+ cell lines based on p-H2AX induction. Application of this assay to primary AML samples identified heterogeneous patterns of repair activity including muted or proficient activation of NHEJ and HRR pathways and predominant activation of NHEJ in a subset of samples. CONCLUSIONS: SCNP identified functional DDR readouts in both NHEJ and HRR pathways, which can be applied to identify cells with BRCA1+/- haploinsuffiency and characterize differential DDR pathway functionality in primary clinical samples.
Subject(s)
DNA Damage , DNA Repair , Single-Cell Analysis/methods , Adult , Ataxia Telangiectasia Mutated Proteins/metabolism , BRCA1 Protein/metabolism , BRCA2 Protein/metabolism , Cell Cycle/drug effects , Cell Line, Tumor , Child , Cyclin A2/metabolism , DNA Breaks, Double-Stranded/drug effects , DNA Repair/drug effects , Dacarbazine/analogs & derivatives , Dacarbazine/pharmacology , Enzyme Inhibitors/pharmacology , Etoposide/pharmacology , Haploinsufficiency/drug effects , Histones/metabolism , Homologous Recombination/drug effects , Humans , Mutagens/toxicity , Phosphorylation/drug effects , Poly(ADP-ribose) Polymerase Inhibitors , Poly(ADP-ribose) Polymerases/metabolism , Reproducibility of Results , TemozolomideABSTRACT
Gemtuzumab ozogamicin (GO), an immunoconjugate between an anti-CD33 antibody and a calicheamicin-γ(1) derivative, induces remissions and improves survival in a subset of patients with acute myeloid leukemia (AML). As the mechanisms underlying GO and calicheamicin-γ(1) resistance are incompletely understood, we herein used flow cytometry-based single cell network profiling (SCNP) assays to study cellular responses of primary human AML cells to GO. Our data indicate that the extent of DNA damage is quantitatively impacted by CD33 expression and drug efflux activity. However, although DNA damage is required for GO-induced cytotoxicity, it is not sufficient for effective cell kill, suggesting that downstream anti-apoptotic pathways may function as relevant resistance mechanisms. Supporting this notion, we found activated PI3K/AKT signaling to be associated with GO resistance in vitro in primary AML cells. Consistently, the investigational AKT inhibitor MK-2206 significantly sensitized various human AML cells to GO or free calicheamicin-γ(1) with particularly pronounced effects in otherwise GO or free calicheamicin-γ(1)-resistant cells. Likewise, MK-2206 also sensitized primary AML cells to calicheamicin-γ(1). Together, our findings illustrate the capacity of SCNP assays to discover chemotherapy-related biological pathways and signaling networks relevant to GO-induced genotoxic stress. The identification of AKT signaling as being associated with GO resistance in vitro may provide a novel approach to improve the in vivo efficacy of GO/calicheamicin-γ(1) and, by extrapolation, other DNA damage-based therapeutics.
Subject(s)
Aminoglycosides/pharmacology , Antibodies, Monoclonal, Humanized/pharmacology , Antineoplastic Agents/pharmacology , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/therapy , Proto-Oncogene Proteins c-akt/physiology , Aminoglycosides/therapeutic use , Antibodies, Monoclonal, Humanized/therapeutic use , Cell Line, Tumor , DNA Damage , Drug Resistance, Neoplasm , Enediynes/pharmacology , Gemtuzumab , Heterocyclic Compounds, 3-Ring/pharmacology , Humans , Phosphatidylinositol 3-Kinases/metabolism , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Sialic Acid Binding Ig-like Lectin 3/metabolism , Signal Transduction/drug effects , Signal Transduction/physiology , Single-Cell Analysis , Tumor Cells, CulturedABSTRACT
This study uses single cell network profiling (SCNP) to characterize biological pathways associated with in vitro resistance or sensitivity to chemotherapeutics commonly used in acute myeloid leukemia (AML) (i.e. cytarabine/daunorubicin, gemtuzumab ozogamicin (GO), decitabine, azacitidine, clofarabine). Simultaneous measurements at the single cell level of changes in DNA damage, apoptosis and signaling pathway responses in AML blasts incubated in vitro with the above drugs showed distinct profiles for each sample and mechanistically different profiles between distinct classes of agents. Studies are ongoing to assess the clinical predictive value of these findings.
